Skip to main content
SpringerLink
Log in
Book cover

Chromatin Remodeling pp 103–113Cite as

Generation of DNA Circles in Yeast by Inducible Site-Specific Recombination

  • Protocol
  • First Online:

  • 3194 Accesses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 833))

Abstract

Site-specific recombinases have been harnessed for a variety of genetic manipulations involving the gain, loss, or rearrangement of genomic DNA in a variety of organisms. The enzymes have been further exploited in the model eukaryote Saccharomyces cerevisiae for mechanistic studies involving chromosomal context. In these cases, a chromosomal element of interest is converted into a DNA circle within living cells, thereby uncoupling the element from neighboring regulatory sequences, obligatory chromosomal events, and other context-dependent effects that could alter or mask intrinsic functions of the element. In this chapter, I discuss general considerations in using site-specific recombination to create DNA circles in yeast and the specific application of the R recombinase.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Raghuraman MK, Brewer BJ, Fangman WL (1997) Cell cycle-dependent establishment of a late replication program. Science 276: 806–809.

    Article  PubMed  CAS  Google Scholar 

  2. Megee PC, Koshland D (1999) A functional assay for centromere-associated sister chromatid cohesion. Science 285: 254–257.

    Article  PubMed  CAS  Google Scholar 

  3. Holmes SG, Broach JR (1996) Silencers are required for inheritence of the repressed state in yeast. Genes Dev 10: 1021–1032.

    Article  PubMed  CAS  Google Scholar 

  4. Bi X, Broach JR (1997) DNA in transcriptionally silent chromatin assumes a distinct topology that is sensitive to cell cycle progression. Mol Cell Biol 17: 7077–7087.

    PubMed  CAS  Google Scholar 

  5. Cheng T-H, Li Y-C, Gartenberg MR (1998) Persistence of an alternate chromatin structure at silenced loci in the absence of silencers. Proc Natl Acad Sci USA 95: 5521–5526.

    Article  PubMed  CAS  Google Scholar 

  6. Cheng T-H, Gartenberg MR (2000) Yeast heterochromatin is a dynamic structure that requires silencers continuously. Genes Dev 14: 452–463.

    PubMed  CAS  Google Scholar 

  7. Kirchmaier AL, Rine J (2001) DNA replication-independent silencing in S. cerevisiae. Science 291: 646–650.

    Article  PubMed  CAS  Google Scholar 

  8. Li Y-C, Cheng T-H, Gartenberg MR (2001) Establishment of transcriptional silencing in the absence of DNA replication. Science 291: 650–653.

    Article  PubMed  CAS  Google Scholar 

  9. Gartenberg MR, Neumann FN, Laroche T, Blaszczyk M, Gasser SM (2004) Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119: 955–967.

    Article  PubMed  CAS  Google Scholar 

  10. Wu CS, Chen YF, Gartenberg MR (2011) Targeted sister chromatid cohesion by Sir2. PLoS Genet 7: e1002000.

    Article  PubMed  CAS  Google Scholar 

  11. Ansari A, Cheng T-H, Gartenberg MR (1999) Isolation of selected chromatin fragments from yeast by site-specific recombination in vivo. Methods 17: 104–111.

    Article  PubMed  CAS  Google Scholar 

  12. Boeger H, Griesenbeck J, Strattan JS, Kornberg RD (2003) Nucleosomes unfold completely at a transcriptionally active promoter. Mol Cell 11: 1587–1598.

    Article  PubMed  CAS  Google Scholar 

  13. Griesenbeck J, Boeger H, Strattan JS, Kornberg RD (2004) Purification of defined chromosomal domains. Methods Enzymol 375: 170–178.

    Article  PubMed  CAS  Google Scholar 

  14. Branda CS, Dymecki SM (2004) Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6: 7–28.

    Article  PubMed  CAS  Google Scholar 

  15. Bischof J, Basler K (2008) Recombinases and their use in gene activation, gene inactivation, and transgenesis. Methods Mol Biol 420: 175–195.

    Article  PubMed  CAS  Google Scholar 

  16. Logie C, Stewart AF (1995) Ligand-regulated site-specific recombination. Proc Natl Acad Sci USA 92: 5940–5944.

    Article  PubMed  CAS  Google Scholar 

  17. Cheng T-H, Chang C-R, Joy P, Yablok S, Gartenberg MR (2000) Controlling gene expression in yeast by inducible site-specific recombination. Nucleic Acids Res 28: E108.

    Article  PubMed  CAS  Google Scholar 

  18. Verzijlbergen KF, Menendez-Benito V, van Welsem T, van Deventer SJ, Lindstrom DL, et al. (2010) Recombination-induced tag exchange to track old and new proteins. Proc Natl Acad Sci USA 107: 64–68.

    Article  PubMed  CAS  Google Scholar 

  19. Tsalik EL, Gartenberg MR (1998) Curing Saccharomyces cerevisiae of the 2 Micron plasmid by targeted DNA damage. Yeast 14: 847–852.

    Article  PubMed  CAS  Google Scholar 

  20. Ghosh SK, Hajra S, Paek A, Jayaram M (2006) Mechanisms for chromosome and plasmid segregation. Annu Rev Biochem 75: 211–241.

    Article  PubMed  CAS  Google Scholar 

  21. Matsuzaki H, Nakajima R, Nishiyama J, Araki H, Oshima Y (1990) Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid. J Bact 172: 610–618.

    PubMed  CAS  Google Scholar 

  22. Gartenberg MR, Wang JC (1993) Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase. Proc Natl Acad Sci USA 90: 10514–10518.

    Article  PubMed  CAS  Google Scholar 

  23. Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15: 1541–1553.

    Article  PubMed  CAS  Google Scholar 

  24. Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) New efficient gene disruption cassette for repeated use in budding yeast. Nucl Acids Res 24: 2519–2524.

    Article  PubMed  Google Scholar 

  25. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5’-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197: 345–346.

    Article  PubMed  CAS  Google Scholar 

  26. Reid RJ, Lisby M, Rothstein R (2002) Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350: 258–277.

    Article  PubMed  CAS  Google Scholar 

  27. Ansari A, Gartenberg MR (1999) Persistence of an alternate chromatin structure at silenced loci in vitro. Proc Natl Acad Sci USA 96: 343–348.

    Article  PubMed  CAS  Google Scholar 

  28. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, et al., editors (2010) Current Protocols in Molecular Biology. New York: John Wiley & Sons.

    Google Scholar 

  29. Smith MC, Brown WR, McEwan AR, Rowley PA (2010) Site-specific recombination by phiC31 integrase and other large serine recombinases. Biochem Soc Trans 38: 388–394.

    Article  PubMed  CAS  Google Scholar 

  30. Weber SA, Gerton JL, Polancic JE, DeRisi JL, Koshland D, et al. (2004) The kinetochore is an enhancer of pericentric cohesin binding. PLoS Biol 2: E260.

    Article  PubMed  Google Scholar 

  31. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115–132.

    Article  PubMed  CAS  Google Scholar 

  32. Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194: 281–301.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA

    Marc R. Gartenberg

  2. The Cancer Institute of New Jersey, New Brunswick, NJ, USA

    Marc R. Gartenberg

Authors
  1. Marc R. Gartenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marc R. Gartenberg .

Editor information

Editors and Affiliations

  1. , Wadsworth Center, New York State Department of Health, New Scotland Avenue 120, Albany, 12208, New York, USA

    Randall H. Morse

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Gartenberg, M.R. (2012). Generation of DNA Circles in Yeast by Inducible Site-Specific Recombination. In: Morse, R. (eds) Chromatin Remodeling. Methods in Molecular Biology, vol 833. Humana Press. https://doi.org/10.1007/978-1-61779-477-3_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-477-3_7

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-476-6

  • Online ISBN: 978-1-61779-477-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation